N-phosphorylaminoethyl phosphate

ABSTRACT

N-phosphorylaminoethyl phosphate is made from phosphoric acid and 2-aminoethanol. It is useful as a therapeutic agent for producing high absorption and retention of phosphate and calcium with extremely low renal calcinosis.

United States Patent Laakso 51 Oct. 10, 1972 l 541N-PHOSPHORYLAMINOETHYL PHOSPHATE I 72 j Inventor: Perttu V. Laakso,Barrington, Ill.

[73] Assignee: The Kendall Company,

Mass.

1221 Filed: Aug. 15,1969

1211 Appl.No.: 850,636

Boston,

[52] US. Cl ....260/926, 424/204 [51} Int. Cl. ..C07f 9/08, C07f 9/22[58] Field of Search ..260/502.5, 926

I 56 References Cited UNITED STATES PATENTS 7/ I 963 Cherbuliez et al..260/247 9/1964 Young et a1. ..260/403 1/1956 Ferrari et al. ..260/944X 6/ l 970 Coleman ..260/926 FOREIGN PATENTS OR APPLICATIONS 931,146 7/1963 Great Britain ..260/926 43/ 1 7,562 7/1967 Japan ..260/926 OTHERPUBLICATIONS Hackh s Chemical Dictionary, McGraw- HilL New York, FourthEdition (1969) page 516.

Primary Examiner-Joseph Rebold Assistant Examiner-Richard L. RaymondAttorney-R. W. Furlong [5 7] ABSTRACT 1 Claim, No DrawingsN-PHOSPHORYLAMINOETHYL PHOSPHATE This invention relates toN-phosphorylaminoethyl phosphate and its use as a therapeutic agent forintroducing phosphate into the bodies of warm-blooded animals.

There have been employed for introducing phosphate into the systems ofwarm-blooded animals in order to adjust the calcium-phosphorus ratio ina variety of phosphate compounds; dosing with these compounds has beenemployed for treatment of hypercalcemia and osteoporosis, inter alia,and has been described by Tisdall and Harris, Journal American MedicalAssociation, Volume 19, 884-887 (1922); Goldsmith and lngbar, Journal ofClinical Investigation, Volume 114, 1053 (1965); Cattaneo andRocchietta, Minerva Medica, Volume 57, 3397-3405 (1966); and Nieper etal. Canadian Pat. No. 760,469.

All of the phosphate therapeutic agents hitherto tried have had variousdisadvantageous characteristics such as bad taste, or have producedunwanted side effects such as diarrhea, renal calcification, or increasein the sodium or potassium ionic level in the body to an undesirablygreat extent.

lt has now been found that a new compound, N- phosphorylaminoethylphosphate is useful as a therapeutic agent for producing both highabsorption and high retention of phosphate (and calcium) together withextremely low renal calcinosis as shown by tests on rats. The compoundis useful in oral dosage form in amounts from 0.3600.036 g. daily, perkilogram of body weight approximately 0.072 g. per kg. being preferredand is effective with diets which are low in calcium as well as withthose containing the normal amount of calcium. The compound is ofrelatively low toxicity, the LD in mice being 1.95 g. per kilogram andthat in rats being 1.5 g. per kilogram. It may be mixed with food or maybe administered in capsule or tablet form. It can be tabletted withconventional tabletting materials such as starch; because of itshygroscopicity it is preferably stored in sealed containers.

The compound N-phosphorylaminoethyl phosphate has the structure:

as verified by elemental analysis, infrared and nuclear magneticresonance spectroscopy, and sodium hydroxide titer.

The following specific example is intended to illustrate more fully thenature of the present invention without serving as a limitation upon itsscope.

EXAMPLE 1 There is placed in a three liter resin kettle fitted with athermometer, dropping funnel, condenser, and magnetic stirrer 2080 g. of85 percent phosphoric acid (18.10 moles). There is placed in thedropping funnel 553 g. of 2-aminoethanol (9.05 moles). The aminoethanolis allowed to flow into the kettle very slowly with continuous stirring,the rate of introduction being such that the temperature rises to about8090 C. and remains within this range. After all of the 2- aminoethanolhas been introduced, the kettle is connected to a vacuum pump and all ofthe free water is distilled off at C. and 0.1 ml. of mercury pressure.At this point the residue in the kettle weighs 2329 g. The temperatureof the reaction mixture is then raised gradually to 180-190 C., andadditional water is distilled off until a total of 632 g. has beencollected. The residue in the flask weighing 2000 g. is a brownsemisolid glassy material. Elemental analysis shows it to have thefollowing composition: carbon 10.87 percent; hydrogen 4.27 percent;nitrogen 5.98 percent; phosphorus 27.85 percent. The material is stablewhen stored under anhydrous conditions, but it is hygroscop ic and inwater it hydrolyzes rapidly to a mixture of aminoethyl phosphate andphosphoric acid, the rate of hydrolysis at C. being approximately 28percent per hour at pH 0.8 and approximately 6 percent per hour at pH7.2. In acid solution the aminoethyl phosphate which is one of the firststage products of the hydrolysis undergoes further hydrolysis tophosphoric acid and 2-aminoethanol at the rate of approximately 2percent per hour at 100 C. at pH 2.7. This further hydrolysis ofaminoethyl phosphate does not occur in alkaline solution.

N-phosphorylaminoethyl phosphate is miscible with water in allproportions but is insoluble in non-polar solvents as well as in suchcommon organic solvents as alcohols, acetone, and ether glycols. Anaqueous solution at 1 percent concentration has a pH of 1.9.

The infrared spectrum of the compound contains the following main peaks(in cm. determined by attenuated total reflection method) 2900, 2300,2200, 2050, 1850,1690,l610,1510,1460,1320,1150,1060, 860, and 800. Theproduct gives a blue color with ninhydrin reagent. Assay by sodiumhydroxide titration gives a purity of 100 percent (98-103 percent).

The nuclear magnetic resonance spectrum of the compound has thefollowing principal peaks: 2.85, 3.33, 3.75, 4.30, and 5.05 ppm relativeto 60 me. at room temperature using deuterium oxide as solvent.

In order to determine the effectiveness of the new compound as atherapeutic agent, it was mixed in measured proportions with a lowphosphorus rat food of the following composition:

Analysis of the foregoing diet showed it to contain 0.32 percent calciumand 0.04 percent phosphorus by weight. To it was added enough of the newcompound to increase the phosphorus content to 0.95 percent. The dietcontaining the added compound was fed to a group of six rats for aperiod of 2 weeks, during which time the amount of daily foodconsumption of each rat was measured, and the amount of calcium andphosphorus in the feces and urine of each rat was determined. Theabsorption was determined by subtracting from the intake the amountexcreted in feces. The retention was calculated by subtracting from theintake the total amount excreted in both feces and urine.

Using the above diet inwhich the per .cent calcium is 0.32 and the percent phosphorus is 0.95, giving a calcium:phosphorus ratio of 1:3.0, theper cent absorption for calcium and phosphorus respectively is 86.8 and98.3, and the per cent retention is 85.2 and 63.3. After the feedingperiod, the rats were sacrificed and the calcium and phosphorus contentof the kidney was determined both by chemical analysis andhistologically by calculating the number of calcium chloranilatecrystals in a cross-sectional area near the end of the kidney. Bothprocedures showed a calcinosis value less than half as great asthat forother phosphate esters observed

